Bladder Carcinogenesis Research Articles

Suppressive Effect of Matrine on Tumor Invasion in N-Butyl-N-(4-Hydroxybutyl)Nitrosamine-Induced Urinary Bladder Carcinogenesis

Aims: This study was designed to investigate the mechanisms and suppressive effects of matrine on the development of urinary bladder cancers induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in rats. Methods: Male Sprague-Dawley rats were given BBN (200 mg/rat) twice a week for a period of 8 weeks. Oral administration of matrine (50 and 100 mg/kg) was started 1 week before BBN exposure for 35 weeks. Half of each bladder was histopathologically analyzed and the remainder was extracted for protein analysis by Western blot. Results: The bladders of BBN-treated rats demonstrated progression from epithelial hyperplasia to papillary urothelial neoplasia and even poorly differentiated invasive cancer. Matrine (50 and 100 mg/kg) treatment decreased the formation of large bladder tumors by 31.6 and 21.1%, respectively. An incidence of cancer cells was detected in rats given BBN [70% (14/20)] and matrine [50 mg/kg: 68.4% (13/19) and 100 mg/kg: 57.9% (11/19), respectively]. The frequency of invasive tumors in the matrine treatment groups [50 mg/kg: 15.4% (2/13), 100 mg/kg: 9.1% (1/11)] was significantly lower than in the BBN-alone group [57% (8/14)]. Furthermore, oral administration of matrine (50 and 100 mg/kg) markedly attenuated the BBN-induced upregulation of bladder cyclooxygenase-2 (COX-2) expression and the elevation of bladder cytosolic phospholipase A2 (cPLA2) levels. Although the contents of 15-prostaglandin dehydrogenase (PGDH), which degrades PGE₂, were dramatically reduced by BBN, matrine exerted no effects on reduced PGDH contents. Conclusion: Our results suggest that matrine suppressed bladder tumor invasion in a rat model, and this might be primarily mediated through regulation of the protein contents, COX-2 and cPLA2 in the bladder.

The distribution in tissues and urine of arsenic metabolites after subchronic exposure to dimethylarsinic acid (DMAV) in rats.

Dimethylarsinic acid (DMA(V)) acted as cancer promoter promoted urinary bladder, liver, and lung carcinogenesis in rats. Understanding of the distribution of arsenicals in critical sites will aid to define the action of DMA(V)-induced toxicity and carcinogenicity. The present experiment was conducted to compare the accumulated levels of arsenicals in the liver, kidney, and bladder of both male and female rats after subchronic exposure to DMA(V). After exposure to DMA(V) in drinking water for 10 weeks, urinary DMA concentrations of 100 and 200 ppm DMA(V)-treated rats increased significantly compared with those of the control rats. Smaller amount of trimethylarsinic acid (TMA) was detected in urine, but not in liver, kidney, and bladder muscle. In the liver and kidney, the levels of DMA in DMA(V)-treated rats significantly increased compared with those of the control group, but there was no difference between 100 and 200 ppm DMA(V)-treated rats. DMA did not accumulate in bladder muscle. There was no difference for DMA concentrations between male and female rats. Our results suggest that the accumulation of DMA in the liver and kidney was saturated above 100 ppm DMA(V) treatment concentration, and DMA(V) was a little partly metabolized to TMA, and TMA was rapidly excreted into urine.

The impact of nutrition in urogenital cancers.

Prostate, bladder and kidney cancers remain the most common cancers of the urinary tract. Despite improved primary prevention, detection and treatment, the incidence of age-related cancers of the urinary tract is likely to rise as a result of global population ageing. An association of diet with prostate, bladder and kidney carcinogenesis is plausible since the majority of metabolites, including carcinogens, are excreted through the urinary tract. Moreover, large regional differences in incidence rates of urologic tumours exist throughout the world. These rates change when people relocate to different geographic areas, which is suggestive of a strong environmental influence. As a result of these observations, numerous studies have been conducted to assess the effects of diet and nutritional status in kidney, bladder and prostate carcinogenesis. Here, we review the literature assessing the effect of diet and nutritional status on urological cancer risk, which has attracted the most interest.

Involvement of the Androgen and Glucocorticoid Receptors in Bladder Cancer.

Bladder cancer is encountered worldwide having been associated with a host of environmental and lifestyle risk factors. The disease has a male to female prevalence of 3 : 1. This disparity has raised the possibility of the androgen receptor (AR) pathway being involved in the genesis of the disease; indeed, research has shown that AR is involved in and is likely a driver of bladder cancer. Similarly, an inflammatory response has been implicated as a major player in bladder carcinogenesis. Consistent with this concept, recent work on anti-inflammatory glucocorticoid signaling points to a pathway that may impact bladder cancer. The glucocorticoid receptor- (GR-) α isoform has an important role in suppressing inflammatory processes, which may be attenuated by AR in the development of bladder cancer. In addition, a GR isoform that is inhibitory to GRα, GRβ, is proinflammatory and has been shown to induce cancer growth. In this paper, we review the evidence of inflammatory mediators and the relationship of AR and GR isoforms as they relate to the propensity for bladder cancer.

Roles of Leucine and Isoleucine in Experimental Models of Bladder Carcinogenesis

Roles of Leucine and Isoleucine in Experimental Models of Bladder Carcinogenesis

Epigenetic inactivation of ST6GAL1 in human bladder cancer.

BackgroundPosttranslational protein modifications are known to modulate key biological processes like proliferation and apoptosis. Accumulating evidence shows that ST6GAL1, an enzyme that catalyzes the transfer of sialic acid onto galactose-containing substrates, is aberrantly expressed in various cancers and may affect cell motility and invasion. This is the first study to describe ST6GAL1 expression and regulation in human bladder cancer.MethodsST6GAL1 mRNA expression levels in human cell lines (UROtsa, RT4, RT112 and J82) and tissue samples (n = 15 normal urothelium (NU), n = 13 papillary non-invasive tumors (pTa), n = 12 carcinoma in situ (CIS), n = 26 muscle invasive tumors (pT2-4)) were assessed using real-time PCR. In addition, ST6GAL1 protein expression was evaluated using immunohistochemistry. Promoter methylation analysis was performed using methylation-specific PCR (MSP) in cell lines (n = 4) and patient samples (n = 23 NU, n = 12 CIS, n = 29 pTa, n = 41 pT2-4). Epigenetic ST6GAL1 gene silencing was confirmed by in vitro demethylation of bladder cell lines. Data were validated by analysis of an independent bladder tumor data set (n = 184) based on The Cancer Genome Atlas (TCGA) portal.ResultsSemi-quantitative ST6GAL1 real-time PCR expression analysis showed two distinct trends: In muscle-invasive tumors ST6GAL1 expression was downregulation by 2.7-fold, while papillary non-invasive tumors showed an increased ST6GAL1 mRNA expression compared to normal urothelium. ST6GAL1 loss in muscle-invasive tumors was associated with increasing invasiveness. On the protein level, 69.2% (n = 45/65) of all tumors showed a weak ST6GAL1 protein staining (IRS ≤ 4) while 25.6% (16/65) exhibited a complete loss (IRS = 0) of ST6GAL1 protein. Tumor-specific DNA methylation of the ST6GAL1 promoter region was frequently found in pT2-4 tumors (53.6% (22/41)), whereas only 13.8% (4/29) of pTa tumors showed ST6GAL1 promoter methylation. Normal urothelium remained unmethylated. Importantly, we significantly revealed an inverse correlation between ST6GAL1 mRNA expression and ST6GAL1 promoter merthylation in primary bladder cancer. These findings were clearly verified by the TCGA public data set and in vitro demethylation assays functionally confirmed ST6GAL1 promoter methylation as a potential regulatory factor for ST6GAL1 gene silencing.ConclusionsOur study characterizes for the first time ST6GAL1 expression loss caused by aberrant ST6GAL1 promoter methylation potentially indicating a tumor suppressive role in bladder carcinogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-901) contains supplementary material, which is available to authorized users.

LINE-1 methylation in granulocyte DNA and trihalomethane exposure is associated with bladder cancer risk

DNA methylation changes contribute to bladder carcinogenesis. Trihalomethanes (THM), a class of disinfection by-products, are associated with increased urothelial bladder cancer (UBC) risk. THM exposure in animal models produces DNA hypomethylation. We evaluated the relationship of LINE-1 5-methylcytosine levels (LINE-1%5mC) as outcome of long-term THM exposure among controls and as an effect modifier in the association between THM exposure and UBC risk. We used a case-control study of UBC conducted in Spain. We obtained personal lifetime residential THM levels and measured LINE-1%5mC by pyrosequencing in granulocyte DNA from blood samples in 548 incident cases and 559 hospital controls. Two LINE-1%5mC clusters (above and below 64%) were identified through unsupervised hierarchical cluster analysis. The association between THM levels and LINE-1%5mC was evaluated with β regression analyses and logistic regression was used to estimate odds ratios (OR) adjusting for covariables. LINE-1%5mC change between percentiles 75th and 25th of THM levels was 1.8% (95% confidence interval (CI): 0.1, 3.4%) among controls. THM levels above vs. below the median (26 μg/L) were associated with increased UBC risk, OR = 1.86 (95% CI: 1.25, 2.75), overall and among subjects with low levels of LINE-1%5mC (n = 975), OR = 2.14 (95% CI: 1.39, 3.30), but not associated with UBC risk among subjects’ high levels of LINE-1%5mC (n = 162), interaction P = 0.03. Results suggest a positive association between LINE-1%5mC and THM levels among controls, and LINE-1%5mC status may modify the association between UBC risk and THM exposure. Because reverse causation and chance cannot be ruled out, confirmation studies are warranted.

Inhibition of bladder cancer invasion by Sp1-mediated BTG2 expression via inhibition of DNA methyltransferase 1.

Significantly lower endogenous expression of B-cell translocation gene 2 (BTG2) was observed in human muscle-invasive bladder cancers (MIBC) than matched normal tissues and non-muscle invasive bladder cancers (NMIBC). BTG2 expression was inversely correlated with increased expression of the DNA methyltransferases DNMT1 and DNMT3a in MIBC, but not NMIBC, suggesting a potential role for BTG2 expression in muscle invasion of bladder cancer. Over 90% of tumor tissues revealed strong methylation at CpG islands of the BTG2 gene, compared with no methylation in the normal tissues, implying epigenetic regulation of BTG2 expression in bladder carcinogenesis. By using EJ bladder cancer cells and the demethylating agent decitabine, transcription of BTG2 was shown to be up-regulated by inhibiting DNMT1 expression via modification at CpG islands. DNMT1 binding to the BTG2 gene further regulated BTG2 expression by chromatin remodeling, such as H3K9 dimethylation and H3K4 trimethylation, and Sp1 activation. Induced BTG2 expression significantly reduced EJ cell tumorigenesis and invasiveness together with induction of G2 /M arrest. These results demonstrate an important role for the BTG2(/TIS21/PC3) gene in the progression of bladder cancers, and suggest that BTG2(/TIS21/PC3) is a promising epigenetic target for prevention of muscle invasion in human bladder cancers.

Clinical significance of the reduction of UT-B expression in urothelial carcinoma of the bladder

Urea transporter B (UT-B) is a membrane protein and plays an important role in regulating urea concentration in bladder urothelial cells. It has been reported that UT-B gene mutations were related to bladder carcinogenesis, and UT-B deletion could induce DNA damage and apoptosis in bladder urothelium. However, the functions and clinical significance of UT-B in human bladder cancer remain unknown. The most common type of bladder cancer is urothelial carcinoma (UC). We hypothesized that UT-B expression was related to bladder UC progress. In this study, UT-B was detected using immunohistochemistry in 52 paraffin-embedded specimens of bladder UC and 10 normal urothelium specimens. The results showed that UT-B protein expression in UC tumor cells was significantly lower as compared with normal urothelial cells (P=0.021). UT-B protein expression was significantly reduced with increasing histological grade (P=0.010). UT-B protein expression in muscle-invasive stage was significantly lower than in non-muscle-invasive stage (P=0.014). Taken together, our data suggest that the reduction or loss of UT-B expression may be related to the incidence, progression and invasiveness of bladder UC. UT-B may be a novel diagnostic or prognostic biomarker, as well as a potential therapeutic target in UC of the bladder.

Abstract 5256: Mechanism of field cancerization

Abstract We developed a strategy that combines histologic and genetic mapping that permits interrogation of the chronology of genetic changes associated with cancer development on a whole-organ scale. By using this approach, we analyzed the sequence of genetic alterations contiguous to the tumor suppressor RB1 and identified a set of alternative target genes that we term “forerunner” (FR) genes whose silencing was associated with development of clonal plaque-like mucosal field effects initiating bladder carcinogenesis. Expression and methylation studies identified five candidate FR genes (ITM2B, LPAR6, MLNR, CAB39L, and ARL11). In vitro mechanistic studies demonstrated that three of these genes (ITM2B, LPAR6 and ARL11) control cell survival and proliferation consistent with their loss of function being contributory to tumorigenesis, and in vivo genetic ablation of Lpar6 in a mouse model resulted in activation of basal/stem cell transcriptional programs involving STAT3 and NFκB causing proliferative expansion of the basal layer of the urothelium. Whole genome analyses of the field effects in a representative whole-organ map revealed a sequential accumulation of alterations in RHO/RAC/Cdc42 pathways that control invasion and cell motility. These studies identify FR genetic alterations in tissue fields as one of the earliest events in carcinogenesis and provide comprehensive description of field cancerization in a primary human tumor. The results have important implications for the development of novel detection markers and chemoprevention therapeutic strategies. Citation Format: Bogdan A. Czerniak, Sangkyou Lee, Jolanta Bondaruk, Sooyong Lee, Tadeusz Majewski, Shizhen Zhang, Li Shen, Yuexin Liu, Charles Guo, Colin Dinney, H. Barton Grossman, Wei Zhang, Menashe Bar-Eli, Keith Baggerly, Richard Behringer, David McConkey, David McConkey, Margaret Knowles, Woonbok Chung, Jean-Pierre Issa. Mechanism of field cancerization. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5256. doi:10.1158/1538-7445.AM2014-5256

Abstract 4209: AMPK suppression in bladder tumorigenesis

Abstract Bladder cancer is the fifth most diagnosed cancer in the US. In 2013 there were approximately 72,570 newly diagnosed cases and 15,210 deaths attributing to this disease where the average age of onset for this disease is 65 and remains one of the most expensive cancers to treat due to lifelong surveillance and invasive procedures. Bladder cancer is defined by two distinct pathways which result in either low grade non-invasive or high grade invasive cancer. Although these are marked by distinct mutations, it has been observed that both display mTORC1 activation. This suggests that activated mTORC1 may be an early contributing factor in bladder tumorigenesis. Additionally, rapamycin, an inhibitor of mTORC1, inhibited tumorigenesis in a mouse model of bladder cancer further implicating mTORC1 as an essential pathway in tumorigenesis. To elucidate the mechanism of mTORC1 activation in bladder cancer, AMP-activated protein kinase (AMPK), a negative regulator of mTORC1, was investigated. AMPK is a critical metabolic regulator that suppresses cellular growth in response to metabolic stress. Additionally it has been observed that AMPK activation is suppressed in breast and hepatocellular carcinoma and the AMPKα2 isoform is suppressed in breast and gastric cancer. To investigate whether levels or activation of AMPK are altered in bladder cancer a pilot cohort of adjacent non-tumor and bladder tumor human samples was obtained as was a human bladder cancer tissue array. In both the pilot cohort and the array, the levels of AMPKα1 and AMPKα2 were suppressed at statistically significant levels in low and high grade bladder cancer when compared to adjacent non-tumor tissue. Messenger RNA expression for both isoforms in bladder tumors revealed that AMPKα2 may be selectively suppressed at the mRNA level. However AMPKα1 suppression appeared to be due to translational or post translational regulatory mechanisms. It was also observed that AMPKα1 was located predominately in the cytoplasm while AMPKα2 was located in both the nuclear and cytoplasmic compartments. This suggests that AMPKα isoforms may have distinct roles in the bladder and may be suppressed by different mechanisms. To test the relevance of AMPK in bladder tumorigenesis the mouse BBN bladder carcinogenesis model was employed. BBN was supplemented in the drinking water of wild-type and AMPKα2-/- mice at a concentration of .05% over the course of 20 weeks. AMPKα2-/- mice displayed a significant increase in bladder weight indicating the presence of larger tumors. There was also an increase in KI67 positive cells in the tumors of AMPKα2-/- mice indicating increased proliferation with no appreciable apoptosis observed in any tumors. Ongoing studies in which BBN is supplemented for 16 weeks and then removed for the duration aim to validate the role of AMPKα2 in bladder tumor initiation and/or progression. In summary, loss of AMPK activity, especially AMPKα2, can promote bladder tumorigenesis through increased proliferation via mTORC1 activity. Citation Format: Stavros Kopsiaftis, Kathryn N. Phoenix, Katie L. Sullivan, John A. Taylor, Kevin P. Claffey. AMPK suppression in bladder tumorigenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4209. doi:10.1158/1538-7445.AM2014-4209

Protective Effect of Diindolylmethane against N-Butyl-N-(4-hydroxybutyl) Nitrosamine-induced Bladder Carcinogenesis

Background Carcinogenesis involves three distinguishable stages, namely, initiation, promotion, and progression. Chemoprevention is the use of agents to inhibit, reverse, or retard tumorigenesis at the initiation stage itself. Diindolylmethane (DIM) is one such agent. The precursor of DIM, that is, indole-3-carbinol, has been found to have beneficial effects in the treatment of prostate and breast cancers. This study was designed to evaluate the protective role of DIM in bladder carcinogenesis. N-Butyl-N-(4-hydroxybutyl) nitrosamine (BBN) was used as a carcinogen to study the chemopreventive activity of DIM in bladder carcinogenesis. Methods Male Wistar rats (n = 24) were grouped into control and experimental groups and the study was terminated at the 32nd week. Group I rats were treated with 0.05% BBN for 8 weeks. Group II rats were treated with BBN + DIM [DIM (5 mg/kg) treatment was started 1 week prior to the BBN treatment, and it was administered for 8 weeks]. Group III rats were treated with DIM alone. Group IV rats were treated as control. The activity and levels of drug-metabolizing and liver enzymes were measured by standard spectrophotometric procedures. Tissues were stained with hematoxylin and eosin and subjected to histopathological examination. Results Male albino Wistar rats were divided into four groups of six animals each. Rats in Group I received BBN and served as experimental control. Group II rats were treated with BBN along with DIM for the initial 8-week period. During the same period, Group III rats were treated with DIM alone. Group IV rats served as the healthy control. At the end of 32 weeks, bladder tissues were collected and subjected to histopathological examination. Increase in the activities of cytochrome (CYP) P450, CYPb5, and CYP c reductase, nicotinamide adenine dinucleotide phosphate (reduced):quinone oxidoreductase 1, aspartate aminotransaminase, and alanine aminotransaminase and decrease in the activity of glutathione S-transferase were found in experimental rats when compared with control rats. Results of histopathological examination showed that DIM reduced BBN-induced initiation of carcinogenesis such as hyperplasia. Conclusion The results of this study show that DIM inhibited hyperplasia, a condition that reflects the development of initiation of carcinogenesis in an experimental model of nitrosamine-induced bladder carcinogenesis, and this effect of DIM can be attributed to its antioxidant activity and its ability to modulate xenobiotic enzymes.

Bacteriuria and urinary schistosomiasis in primary school children in rural communities in Enugu State, Nigeria, 2012.

IntroductionAccording to a study conducted in1989, Enugu State has an estimated urinary schistosomiasis prevalence of 79%. Recently, studies have implicated bacteriuria co-infection in bladder cancer. These bacteria accelerate the multi-stage process of bladder carcinogenesis. Knowledge about the prevalence of this co-infection is not available in Enugu and the information provided by the 1989 study is too old to be used for current decision making.MethodsWe carried out a cross-sectional survey of primary school children aged5-15years, who were randomly selected through a multi stage sampling method using guidelines recommended by WHO for schistosomiasis surveys. An interviewer administered questionnaire was used to collect data on demography, socioeconomic variables and clinical presentations. Urine samples were collected between 10.00am and 2.00pm. Each sample was divided into two: (A) for prevalence and intensity using syringe filtration technique and (B) for culture. Intensity was categorized as heavy (>50ova/10mls urine) and light (<50ova/10mls urine). Significant bacteriuria was bacteria count ≥ 105 colony forming units/ml of urine.ResultsOf the 842 pupils, 50.6% were females. The prevalence of urinary schistosomiasis was 34.1%. Infection rate was higher(52.8%) among 13-15 years(Prevalence Ratio = 2.45, 95% Confidence Interval 1.63-3.69). Heavy infections wad 62.7% and egg count/10mls urine ranged from 21-1138. Significant bacteriuria among pupils with urinary schistosomiasis was 53.7% compared to 3.6% in the uninfected(PR = 30.8,95% CI 18.91- 52.09). The commonest implicated organism was Escherchia coli.ConclusionWe found high prevalence of bacteriuria co-infection among children with urinary schistosomiasis in Enugu State. This underscores the need for concurrent antibiotics administration and follow-up to avert later complications.

Downregulation of glutathione S-transferase M1 protein in N-butyl-N-(4-hydroxybutyl)nitrosamine-induced mouse bladder carcinogenesis

Bladder cancer is highly recurrent following specific transurethral resection and intravesical chemotherapy, which has prompted continuing efforts to develop novel therapeutic agents and early-stage diagnostic tools. Specific changes in protein expression can provide a diagnostic marker. In our present study, we investigated changes in protein expression during urothelial carcinogenesis. The carcinogen BBN was used to induce mouse bladder tumor formation. Mouse bladder mucosa proteins were collected and analyzed by 2D electrophoresis from 6 to 20weeks after commencing continuous BBN treatment. By histological examination, the connective layer of the submucosa showed gradual thickening and the number of submucosal capillaries gradually increased after BBN treatment. At 12-weeks after the start of BBN treatment, the urothelia became moderately dysplastic and tumors arose after 20-weeks of treatment. These induced bladder lesions included carcinoma in situ and connective tissue invasive cancer. In protein 2D analysis, the sequentially downregulated proteins from 6 to 20weeks included GSTM1, L-lactate dehydrogenase B chain, keratin 8, keratin 18 and major urinary proteins 2 and 11/8. In contrast, the sequentially upregulated proteins identified were GSTO1, keratin 15 and myosin light polypeptide 6. Western blotting confirmed that GSTM1 and NQO-1 were decreased, while GSTO1 and Sp1 were increased, after BBN treatment. In human bladder cancer cells, 5-aza-2′-deoxycytidine increased the GSTM1 mRNA and protein expression. These data suggest that the downregulation of GSTM1 in the urothelia is a biomarker of bladder carcinogenesis and that this may be mediated by DNA CpG methylation.

Potential chemoprotective effects of green propolis, L-lysine and celecoxib on bone marrow cells and peripheral blood lymphocytes of Wistar rats subjected to bladder chemical carcinogenesis

To evaluate the genotoxicity of propolis and L-lysine, as well as their effects on the possible cellular damage in erythroblasts (bone marrow) and leukocytes (peripheral blood) caused by the carcinogen BBN (n - butyl - n {4 - hydroxybutyl} nitrosamine) in rats subjected to bladder carcinogenesis and treated with green propolis and L-lysine. One hundred and twenty five rats were distributed into the following groups: I, IIA, IIB, III, K, L M N, X, XI, XII and XIII. Groups I to X received BBN in drinking water for 14 weeks (wks). Group I was treated with intragastric (ig) propolis at 150 mg/kg body weight, for 44 wks, beginning 30 days before start of BBN. Groups IIA and III were treated with propolis (150 mg/kg), for 40 wks, subcutaneous (sc) and ig, respectively, beginning simultaneously with BBN. On the 32nd wk, the animals of groups L, M and N were treated ig with L-lysine (300 mg/kg), celecoxib (30 mg/kg) and propolis (300 mg/kg), respectively, up to the 40th wk. The groups that received only BBN (IIB and K) were treated with water, sc and orally, respectively, for 40 wks. Groups XI, XII and XIII received respectively propolis (150 mg/kg), L-lysine (150 mg/kg) and water ig for 40 wks. After 40 wks, the surviving animals were anesthetized and subjected to femoral bone marrow aspiration and blood collection from the aorta, for CA and MNT, respectively, for investigation of genotoxicity. Groups IIB and K, which received only BBN and water, showed the greatest DNA damage in peripheral leukocytes (CA) and largest number of micronuclei in bone marrow erythrocytes (MNT) in relation to all other groups that received BBN and lysine and/or propolis (p<0.001). Both propolis and L-lysine are effective in protecting against genotoxicity, as well not being genotoxic themselves toward the cells evaluated, at the doses and times administered and according to the two tests utilized.

Vascular endothelial growth factor mRNA levels as a biomarker for short-term N-butyl-N-(4-hydroxybutyl) nitrosamine-induced rat bladder carcinogenesis bioassay.

Generically, carcinogenic effects of chemicals in bladder carcinogenesis are judged by induction of papillary or nodular (PN) hyperplasia in rats given N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) for 4 weeks and the test chemical for 22-28 weeks. However, upregulation of vascular endothelial growth factor (VEGF) begins early in rat BBN bladder carcinogenesis. To establish a short-term rat bladder carcinogenic bioassay, we analyzed the correlations between VEGF, VEGF mRNA and bladder lesions inductions at 10 and 26 weeks after BBN treatment. Six-week-old male Wistar (slc) rats were given 0.05% BBN for 4, 10 or 26 weeks. To avoid individual rat bias, the bladders were investigated by partial cystectomy at 10 weeks and total cystectomy at 26 weeks. After induction, PN hyperplasia and carcinoma in rats increased with the length of BBN treatment and immunohistochemical VEGF expression also increased following carcinogenesis, but the immunoreactivity of individual lesions was quite variable. Moreover, induction of PN hyperplasia at 10 weeks' BBN treatment was not significantly correlated with that at 26 weeks' treatment; thus, it was not possible to predict the carcinogenic effect due to the induction of PN hyperplasia at 26 weeks' BBN treatment by that at 10 weeks' treatment. However, VEGF mRNA levels of rat bladders at 10 weeks' BBN treatment revealed a strong significant correlation with the incidence of bladder lesions at 26 weeks' treatment. Here, we suggest that quantitative VEGF mRNA levels are a good biomarker for a short-term BBN-induced bioassay for rat bladder carcinogenesis.

Dimethylarsinic acid (DMA(V) ) changed the expressions of proliferative related factors and secretion of inflammatory cytokines in rat bladder.

Dimethylarsinic acid (DMA(V) ), the major urinary metabolite of inorganic arsenic, is a urinary bladder carcinogen and bladder tumor promoter in adult rats. Increased urothelial cellular proliferation has been considered as an earlier phenotype in DMA(V) -induced bladder carcinogenesis. The present study examined the ultrastructural changes of bladder epithelial cells and expressions of proliferation factors, as well as the secretion of inflammatory cytokines in rats exposed to DMA(V) for 10 weeks by transmission electron microscopy (TEM), qRT-PCR, immunohistochemical staining and ELISA methods. The results showed that DMA(V) administered in the drinking water produced urothelial cytotoxicity and ultrastructural changes in rats. PCNA, cyclin D1 and COX-2 mRNA expressions and immunoreactivities were elevated in bladder urothelium. In addition, 200 ppm DMA(V) treatment increased the transforming growth factor-beta 1 (TGF-β1) secretion and decreased tumor necrosis factor-alpha (TNF)-α level in the urine of rats. These data suggest that chronic inflammation, bladder epithelium lesions and proliferation might be the basic process of the chronic toxicity effects in DMA(V) -treated rats.

Effects of seven chemicals on DNA damage in the rat urinary bladder: A comet assay study

The in vivo comet assay has been used for the evaluation of DNA damage and repair in various tissues of rodents. However, it can give false-positive results due to non-specific DNA damage associated with cell death. In this study, we examined whether the in vivo comet assay can distinguish between genotoxic and non-genotoxic DNA damage in urinary bladder cells, by using the following seven chemicals related to urinary bladder carcinogenesis in rodents: N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), glycidol, 2,2-bis(bromomethyl)-1,3-propanediol (BMP), 2-nitroanisole (2-NA), benzyl isothiocyanate (BITC), uracil, and melamine. BBN, glycidol, BMP, and 2-NA are known to be Ames test-positive and they are expected to produce DNA damage in the absence of cytotoxicity. BITC, uracil, and melamine are Ames test-negative with metabolic activation but have the potential to induce non-specific DNA damage due to cytotoxicity. The test chemicals were administered orally to male Sprague-Dawley rats (five per group) for each of two consecutive days. Urinary bladders were sampled 3h after the second administration and urothelial cells were analyzed by the comet assay and subjected to histopathological examination to evaluate cytotoxicity. In the urinary bladders of rats treated with BBN, glycidol, and BMP, DNA damage was detected. In contrast, 2-NA induced neither DNA damage nor cytotoxicity. The non-genotoxic chemicals (BITC, uracil, and melamine) did not induce DNA damage in the urinary bladders under conditions where some histopathological changes were observed. The results indicate that the comet assay could distinguish between genotoxic and non-genotoxic chemicals and that no false-positive responses were obtained.

Complex relationships between occupation, environment, DNA adducts, genetic polymorphisms and bladder cancer in a case-control study using a structural equation modeling.

DNA adducts are considered an integrate measure of carcinogen exposure and the initial step of carcinogenesis. Their levels in more accessible peripheral blood lymphocytes (PBLs) mirror that in the bladder tissue. In this study we explore whether the formation of PBL DNA adducts may be associated with bladder cancer (BC) risk, and how this relationship is modulated by genetic polymorphisms, environmental and occupational risk factors for BC. These complex interrelationships, including direct and indirect effects of each variable, were appraised using the structural equation modeling (SEM) analysis. Within the framework of a hospital-based case/control study, study population included 199 BC cases and 213 non-cancer controls, all Caucasian males. Data were collected on lifetime smoking, coffee drinking, dietary habits and lifetime occupation, with particular reference to exposure to aromatic amines (AAs) and polycyclic aromatic hydrocarbons (PAHs). No indirect paths were found, disproving hypothesis on association between PBL DNA adducts and BC risk. DNA adducts were instead positively associated with occupational cumulative exposure to AAs (p = 0.028), whereas XRCC1 Arg 399 (p<0.006) was related with a decreased adduct levels, but with no impact on BC risk. Previous findings on increased BC risk by packyears (p<0.001), coffee (p<0.001), cumulative AAs exposure (p = 0.041) and MnSOD (p = 0.009) and a decreased risk by MPO (p<0.008) were also confirmed by SEM analysis. Our results for the first time make evident an association between occupational cumulative exposure to AAs with DNA adducts and BC risk, strengthening the central role of AAs in bladder carcinogenesis. However the lack of an association between PBL DNA adducts and BC risk advises that these snapshot measurements are not representative of relevant exposures. This would envisage new scenarios for biomarker discovery and new challenges such as repeated measurements at different critical life stages.

NOV is upregulated and promotes migration and invasion in bladder cancer

NOV is pro-tumourigenic via epithelial-mesenchymal transition (EMT) in several malignancies but is not studied in bladder cancer (BCa). Whether NOV is responsible for bladder carcinogenesis and the underlying mechanism is unclear. Using immunohistochemical staining, we quantified expressions of NOV, pS6, Vimentin and E-cadherin in 66 bladder cancer and 10 normal bladder urothelium samples. EMT was profiled by EMT index (EMTi) calculated as the ratio of Vimentin to E-cadherin. In vitro and in vivo studies were carried out to profile the role of NOV in the tumourigenesis of BCa. NOV was upregulated in bladder cancer compared to normal tissue, and its expression was correlated to pS6 and EMTi. Expression of NOV was higher in recurrent and multiple tumours and was increased with progression of tumour grade. NOV expression was also higher in BCa cell lines. Silence of NOV attenuated EMT, decreased invasion and migration of BCa cells. Silence of NOV also inhibited xenograft tumour growth and decreased tumour EMT. NOV is pro-tumourigenic in bladder cancer especially in nonmuscle-invasive entities (NMIBC). NOV may promote carcinogenesis via promotion of EMT and association with increased mTOR activity.